Evidence for positive selection and population structure at the human MAO-A gene Yoav Gilad*†, Shai Rosenberg‡, Molly Przeworski§, Doron Lancet*, and Karl Skorecki†‡ *Department of Molecular Genetics and the Crown Human Genome Center, The Weizmann Institute of Science, Rehovot 76100, Israel; ‡Rappaport Faculty of Medicine and Research Institute, Technion–Israel Institute of Technology, and Rambam Medical Center, Haifa 31096, Israel; and §Department of Statistics, University of Oxford, 1 South Parks Road, Oxford OX1 3TG, United Kingdom Communicated by Eviatar Nevo, University of Haifa, Haifa, Israel, November 19, 2001 (received for review May 15, 2001) We report the analysis of human nucleotide diversity at a genetic locus known to be involved in a behavioral phenotype, the mono- amine oxidase A gene. Sequencing of five regions totaling 18.8 kb and spanning 90 kb of the monoamine oxidase A gene was carried out in 56 male individuals from seven different ethnogeographic groups. We uncovered 41 segregating sites, which formed 46 distinct haplotypes. A permutation test detected substantial pop- ulation structure in these samples. Consistent with differentiation between populations, linkage disequilibrium is higher than ex- pected under panmixia, with no evidence of a decay with distance. The extent of linkage disequilibrium is not typical of nuclear loci and suggests that the underlying population structure may have been accentuated by a selective sweep that fixed different hap- lotypes in different populations, or by local adaptation. In support of this suggestion, we find both a reduction in levels of diversity Fig. 1. Overall genomic structure and sequencing strategy for the MAO-A (as measured by a Hudson–Kreitman–Aguade test with the DMD44 gene. The arrangement of exons is shown relative to the scale provided at the locus) and an excess of high frequency-derived variants, as ex- top. We indicate the position of each of the five resequenced regions. The pected after a recent episode of positive selection. sequencing strategy is illustrated for region 2, where the PCR products are shown as overlapping segments. ssociation studies at the monoamine oxidase A (MAO-A) Alocus have been motivated by the striking finding that mutations in the gene result in borderline mental retardation Methods and abnormal behavior, including increased impulsive behavior DNA Samples. Human genomic DNA was derived from two (1). It has been proposed that a broad range of interindividual sources. (i) Thirty three DNA samples were provided by Coriel human variability in related behavioral phenotypes may be Cell Repositories, Camden, NJ. These consisted of: seven associated with nucleotide variation at this locus, in particular Pygmy samples, nine Aboriginal Taiwanese, three Chinese, with the well-documented range of interindividual variability in two Japanese, five Mexicans, and seven Russians. (ii) Samples the activity level of the gene product (2). from 23 unrelated individuals were provided by the National Numerous studies have been carried out on the association of Laboratory for the Genetics of Israeli Populations at Tel Aviv this genetic locus to behavioral phenotypes (3–5), but positive University; these came from two ethnic groups: Ashkenazi and negative associations have been accepted with reservation Jews (13 individuals) and Bedouins (10 individuals). We and have been difficult to replicate in subsequent studies. Some isolated genomic DNA from two common chimpanzees (Pan of these difficulties may result from a failure to take into account troglodytes) from blood kindly provided by Yigal Horvitz of the the evolutionary history of the region, including the effects of Israeli Safari Zoo (Ramat-Gan, Israel), using the Genomix population stratification and͞or of natural selection. These DNA preparation kit (Talent SRL, Trieste, Italy). factors shape the distribution of linkage disequilibrium (LD) and hence the likelihood of an association. Sequencing Strategy. The MAO-A gene spans more than 90 kb. As a step in our understanding of genetic variability at this We chose five segments that varied from 2 to 5 kb in length and locus, we examined polymorphism patterns in normal, unre- totaled 18.8 kb (Fig. 1). We tried to include as much exon lated individuals. We used direct resequencing of the MAO-A sequence as possible while keeping the segments equally distrib- gene. A number of indirect approaches have been used for uted across the entire gene. Overlapping Ϸ1-kb PCR products large-scale single nucleotide polymorphism (SNP) discovery were sequenced across each segment. The sequence we screened and analysis (6, 7). However, direct resequencing is the most consisted of 95.7% introns and 4.3% exons. reliable approach to SNP discovery, affording a complete picture of the sequence variation for a given genomic region. PCR Procedures. We designed specific PCR primers for the To establish the phase of segregating sites across long genomic amplification of the Ϸ1-kb segments of the MAO-A gene, based segments of autosomal loci, previous studies have often in- ferred haplotypes by means of a variety of algorithms (e.g., ref. 8). These have difficulty in reconstructing the phase of SNPs Abbreviations: MAO-A, monoamine oxidase A; SNP, single nucleotide polymorphism; LD, at low frequency. Here, we are able to determine haplotypes linkage disequilibrium. †To whom reprint requests may be addressed. E-mail: [email protected] or directly in males, because MAO-A is sex-linked. The region [email protected]. reported in this study is one of the longest stretches of DNA The publication costs of this article were defrayed in part by page charge payment. This in a recombining part of the genome for which haplotypes have article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. been obtained directly. §1734 solely to indicate this fact. 862–867 ͉ PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.022614799 Downloaded by guest on October 1, 2021 Table 1. Individual haplotypes Haplotypes of 56 males, spanning the 90-kb MAO-A gene. The 41 polymorphic sites are indicated by their segment name (first row) and the position within the segment (second row). The human consensus (frequent allele), human rare allele, and the chimpanzee consensus are indicated in rows 3–5. The rare variant is represented by a dark rectangle with the digit 1. We also indicate the human and chimpanzee consensus sequences. on the available sequences. We performed PCR in a total volume 50 mM KCl, 10 mM Tris (pH 8.3), 1 unit of Taq DNA polymerase GENETICS of 25 ␮l, containing 0.2 mM of each deoxynucleotide (Promega), (Roche Molecular Biochemicals), and 50 ng of genomic DNA. 50 pMol of each primer, PCR buffer containing 1.5 mM MgCl2, PCR conditions were as follows: 35 cycles of denaturation at Gilad et al. PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 ͉ 863 Downloaded by guest on October 1, 2021 94°C, annealing at either 55°Cor52°C, and extension at 72°C, implements the coalescent with recombination. For a given each step for 1 min. The first step of denaturation and the last population recombination rate, simulations were run conditional step of extension were 3 min and 10 min, respectively. PCR on the actual number of base pairs and sample size. The resulting products were separated on a 1% agarose gel to view their size, analysis shows that with recombination the variance of H de- and they were purified by using the High Pure PCR Product creases. Thus, there is a reduction in the proportion of runs in Purification Kit (Roche Molecular Biochemicals). which P Ͻ 0.05, indicating that the test becomes more conser- vative when critical values for no recombination are used. This DNA Sequencing. Sequencing reactions were performed on PCR is demonstrated below by the H test P values reported for products or clones in both directions with dye terminators (dye different rates of recombination. terminator cycle sequencing kit; Perkin–Elmer) on an Applied To test for differentiation between populations, we used the Biosystems 3700 automated sequencer. Snn test (15). This test is based on the idea that, in the presence After base calling with Applied Biosystems ANALYSIS of population structure, the nearest neighbors (in sequence software (version 3.0), the analyzed data were edited by us- space) of a haplotype will tend to be found in the same ing the SEQUENCHER program, version 3.0 (Gene Codes, Ann population as that haplotype more often than they would be Arbor, MI). under panmixia. This test has been shown to be more powerful than ␹2 tests of homogeneity for small samples, especially in the Determination of Polymorphism and Divergence. We sequenced presence of recombination (16). each Ϸ1-kb genomic segment from both ends for each individ- To summarize pairwise LD, we used the common measure, DЈ ual. The SEQUENCHER software was used to assemble the (17), a summary of pairwise LD normalized so that the range sequences and identify DNA polymorphisms. We repeated the would be between Ϫ1 and 1. We used Fisher’s Exact Test to sequencing reaction of any segment originally identified as determine whether the pairs of sites were in significant LD. containing a singleton. The human sequences were aligned with The recombination rate per generation was estimated by using the chimpanzee sequence to identify fixed differences. the approach of Payseur and Nachman (18) (see also http:͞͞ eebweb.arizona.edu͞nachman͞publications͞data͞microsats. Data Analysis. We calculated three summaries of diversity levels: html), which is based on a comparison of a physical map (the ␪ Watterson’s W (9), based on the number of segregating sites in GB4 radiation hybrid map) and the Genethon genetic map (see the sample; ␲ (10), the average number of pairwise differences ref. 18 for details). To estimate for the rate for this locus, we used ␪ in the sample; and H, a summary that gives more weight to high two microsatellites, DXS1201 and DXS1043, in close proximity frequency-derived variants (11).